Protein synthesis modulates the biological effectiveness of the combined action of hyperthermia and high-LET radiation.

The combined effects of heavy-ion radiation and hyperthermia on the survival of CHO-SC1 cells and its temperature-sensitive (ts) mutant tsH1 cells were studied using accelerated neon ions followed by mild heating at 41.5 degrees C. The sequence of application of heat and high-LET radiation is significant to cell-killing effects. Heat applied to cells prior to irradiation with neon plateau ions (LET = 32 keV/microns) was less effective than heat applied immediately after irradiation. The ability of cells to synthesize new proteins plays a key role in this sequence-dependent thermal sensitization. When protein synthesis was shut down in tsH1 cells, the thermal enhancement of cell killing by high-LET radiation was the same regardless of the sequence. The thermal enhancement of radiation-induced cell killing was LET-dependent for the SC1 cells, but this was not clearly demonstrated in the tsH1 cells. Furthermore, the RBE of heated SC1 cells varied with LET and reached a maximum of greater than 3 at 80 keV/microns. In the absence of protein synthesis, the maximum RBE value was reduced to 2.6. These results suggest that the accumulation of cellular damage caused by exposure to densely ionizing particles with increasing LETs can be potentiated with active protein synthesis during postirradiation heat treatment.     

Mutagenic effects of carbon ions near the range end in plants

To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/μm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113keV/μm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/μm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>～30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/μm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/μm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.     

LET and ion species dependence for cell killing in normal human skin fibroblasts

We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.     

Inactivation of aerobic and hypoxic cells from three different cell lines by accelerated (3)He-, (12)C- and (20)Ne-ion beams

The LET-RBE spectra for cell killing for cultured mammalian cells exposed to accelerated heavy ions were investigated to design a spread-out Bragg peak beam for cancer therapy at HIMAC, National Institute of Radiological Sciences, Chiba, prior to clinical trials. Cells that originated from a human salivary gland tumor (HSG cells) as well as V79 and T1 cells were exposed to (3)He-, (12)C- and (20)Ne-ion beams with an LET ranging from approximately 20-600 keV/micrometer under both aerobic and hypoxic conditions. Cell survival curves were fitted by equations from the linear-quadratic model and the target model to obtain survival parameters. RBE, OER, alpha and D(0) were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/micrometer, then decreased with a further increase in LET. Clear splits of the LET-RBE or -OER spectra were found among ion species and/or cell lines. At a given LET, the RBE value for (3)He ions was higher than that for the other ions. The position of the maximum RBE shifts to higher LET values for heavier ions. The OER value was 3 for X rays but started to decrease at an LET of around 50 keV/micrometer, passed below 2 at around 100 keV/micrometer, and then reached a minimum above 300 keV/micrometer, but the values remained greater than 1. The OER was significantly lower for (3)He ions than the others.     

The RBE-LET relationship for rodent intestinal crypt cell survival, testes weight loss, and multicellular spheroid cell survival after heavy-ion irradiation.

This report presents data for survival of mouse intestinal crypt cells, mouse testes weight loss as an indicator of survival of spermatogonial stem cells, and survival of rat 9L spheroid cells after irradiation in the plateau region of unmodified particle beams ranging in mass from 4He to 139La. The LET values range from 1.6 to 953 keV/microns. These studies examine the RBE-LET relationship for two normal tissues and for an in vitro tissue model, multicellular spheroids. When the RBE values are plotted as a function of LET, the resulting curve is characterized by a region in which RBE increases with LET, a peak RBE at an LET value of 100 keV/microns, and a region of decreasing RBE at LETs greater than 100 keV/microns. Inactivation cross sections (sigma) for these three biological systems have been calculated from the exponential terminal slope of the dose-response relationship for each ion. For this determination the dose is expressed as particle fluence and the parameter sigma indicates effect per particle. A plot of sigma versus LET shows that the curve for testes weight loss is shifted to the left, indicating greater radiosensitivity at lower LETs than for crypt cell and spheroid cell survival. The curves for cross section versus LET for all three model systems show similar characteristics with a relatively linear portion below 100 keV/microns and a region of lessened slope in the LET range above 100 keV/microns for testes and spheroids. The data indicate that the effectiveness per particle increases as a function of LET and, to a limited extent, Z, at LET values greater than 100 keV/microns. Previously published results for spread Bragg peaks are also summarized, and they suggest that RBE is dependent on both the LET and the Z of the particle.     

RBE for carcinogenesis following exposure to high LET radiation

Stochastic radiation effects following exposure to heavy ions and other high linear energy transfer (LET) radiation in space are a matter of concern when the long-term consequences of space flights are considered. This paper is an overview of the relevant literature, emphasizing uncertainties entailed from estimates of relative biological effectiveness (RBE) for different experiment end-points, making the choice of a single weighting factor for the prediction of cancer risk in man extremely difficult. Life-span-shortening studies in mice exposed to heavy ions and ongoing large-scale experiments in monkeys exposed to protons suggest that RBEs for all cancers are lower than 5. This does not exclude a much higher RBE for rare tumors such as brain tumors in monkeys or promoted Harderian gland tumours in mice at LET >80 keV/µm. Skin cancer studies in rats exposed to neon or argon resulted in similar RBE. Exposure to fission neutrons led to high RBE in all species, not excluding values much higher than 20 for specific cancers such as lung tumors in mice and all cancers in rats. The estimate of maximal RBE is, however, extremely dependent on the hypothesis made on the shape of the dose-response curves in the lower range of doses. These results suggest that neutrons may be the most hazardous component of high-LET radiation. There is only limited evidence from cancer experiments that LET >150 keV/µm results in highly decreased efficiency, but this has been found for bone cancer induction following exposure to fission fragments.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Accelerated heavy particles and the lens. VI. RBE studies at low doses

We report on the prevalence, hazard, and relative biological effectiveness (RBE) for various stages of lens opacification in rats induced by very low doses of fast argon ions of LET 88 keV/microns, compared to those for X rays. Doses of argon ions from 0.01 to 0.25 Gy were used and RBEs of these ions relative to X rays estimated using a nonparametric technique. At the end of the follow-up period, which encompasses a significant fraction of the animals' lifetime, 90% confidence intervals for the RBE of the argon ions relative to X rays were 4-8 at 0.25 Gy, 10-40 at 0.05 Gy, and 50-100 at 0.01 Gy. Our results are consistent with the point-estimate neutron RBEs in Japanese A-bomb survivors, though broad confidence bounds are present in the Japanese results. If a reasonable extrapolation to higher doses is used, our results are also consistent with data reported earlier at higher doses for argon-ion cataractogenesis in rats, mice, and rabbits. We conclude from these results that at very low doses the RBE for cataractogenesis from HZE particles in space is considerably more than 20, and use of a quality factor of at least 50 would be prudent.     

Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions

The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.     

Heavy ion effects on cellular DNA: strand break induction and repair in cultured diploid lens epithelial cells

The relative biological effectiveness (RBE) for the induction of DNA strand breaks and the efficiency of repair of these breaks in cultured diploid bovine lens epithelial cells was measured, using accelerated heavy ions in the linear energy transfer (LET)-range up to 16,200 keV/micron. At LET values above 800 keV/micron, the number of DNA strand breaks induced per particle increases both with the atomic number of the projectile and with its kinetic energy. About 90 per cent or more of the strand breaks induced by ions with an LET of less than 10,000 keV/micron are repaired within 24 h. Repair kinetics show a dependence on the particle fluence (irradiation dose). At higher particle fluences a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At any LET value, repair is much slower after heavy-ion exposure than after X-irradiation. This is especially true for low energetic particles with a very high local density of energy deposition within the particle track. At the highest LET value (16,200 keV/micron), no significant repair is observed.     

Relative biological effectiveness of high-energy iron ions for micronucleus formation at low doses

Dose-response curves for micronucleus (MN) formation were measured in Chinese hamster V79 and xrs6 (Ku80(-)) cells and in human mammary epithelial MCF10A cells in the dose range of 0.05-1 Gy. The Chinese hamster cells were exposed to 1 GeV/nucleon iron ions, 600 MeV/nucleon iron ions, and 300 MeV/nucleon iron ions (LETs of 151, 176 and 235 keV/microm, respectively) as well as with 320 kVp X rays as reference. Second-order polynomials were fitted to the induction curves, and the initial slopes (the alpha values) were used to calculate RBE. For the repair-proficient V79 cells, the RBE at these low doses increased with LET. The values obtained were 3.1 +/- 0.8 (LET = 151 keV/microm), 4.3 +/- 0.5 (LET = 176 keV/microm), and 5.7 +/- 0.6 (LET = 235 keV/microm), while the RBE was close to 1 for the repair-deficient xrs6 cells regardless of LET. For the MCF10A cells, the RBE was determined for 1 GeV/nucleon iron ions and was found to be 5.5 +/- 0.9, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/nucleon iron-ion beam was intercepted by various thicknesses of high-density polyethylene plastic absorbers, which resulted in energy loss and fragmentation. It was found that the MN yield for V79 cells placed behind the absorbers decreased in proportion to the decrease in dose both before and after the iron-ion Bragg peak, indicating that RBE did not change significantly due to shielding except in the Bragg peak region. At the Bragg peak itself with an entrance dose of 0.5 Gy, where the LET is very high from stopping low-energy iron ions, the effectiveness for MN formation per unit dose was decreased compared to non-Bragg peak areas.     

Effect of inhibitors of DNA synthesis on the sensitivity of mammalian cells to radiation with different levels of linear energy transfer

Radiosensitivity of Chinese hamster cells increased by 1.71 times in the presence of arabinoside cytosine and hydroxyurea after gamma-irradiation, and no sensitization occurred after irradiation with carbon ions of 6.6 MeV/nuclon (LET, 227 keV/micron). Under a standard set of conditions, the RBE coefficient of carbon ions decreased from 3.09 to 1.78 in the presence of DNA synthesis inhibitors. The possible mechanism of this phenomenon is discussed.     

Charged-particle mutagenesis. 1. Cytotoxic and mutagenic effects of high-LET charged iron particles on human skin fibroblasts.

Cytotoxic and mutagenic effects of high-LET charged iron (56Fe) particles were measured quantitatively using primary cultures of human skin fibroblasts. Argon and lanthanum particles and gamma rays were used in comparative studies. The span of LETs selected was from 150 keV/microns (330 MeV/u) to 920 keV/microns (600 MeV/u). Mutations were scored at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus using 6-thio-guanine (6-TG) for selection. Exposure to these high-LET charged particles resulted in exponential survival curves. Mutation induction, however, was fitted by the linear model. The relative biological effectiveness (RBE) for cell killing ranged from 3.7 to 1.3, while that for mutation induction ranged from 5.7 to 0.5. Both the RBE for cell killing and the RBE for mutagenesis decreased with increasing LET over the range of 1.50 to 920 keV/microns. The inactivation cross section (sigma i) and the action cross section for mutation induction (sigma m) ranged from 32.9 to 92.0 microns2 and 1.45 to 5.56 X 10(-3) microns2; the maximum values were obtained by 56Fe with an LET of 200 keV/microns. The mutagenicity (sigma m/sigma i) ranged from 2.05 to 7.99 X 10(-5) with an inverse relationship to LET.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

The relative biological effectiveness of 670 MeV/A neon as a function of depth in water for a tissue model

Linear energy transfer (LET infinity) spectra of identified charge fragments and primaries, produced by nuclear interactions of 670 MeV/A neon in water, were measured along the unmodulated Bragg curve of the neon beam. The relative biological effectiveness (RBE) values for spermatogonial cell killing, as reported on the basis of weight loss assay of mouse testes irradiated with beams of approximately constant single LET infinity, were summed over the particle LET infinity spectra to obtain an effective RBE for each charged-particle species, as a function of water absorber thickness. The resultant values of effective RBE were combined to obtain an effective RBE for the mixed radiation field. The RBE calculated in this way was compared with experimental RBEs obtained for spermatogonial cell killing in the mixed radiation field produced by neon ions traversing a thick water absorber. Discrepancies of 10-40% were observed between the calculated RBE and the RBE measured in the mixed radiation field. Part of this discrepancy can be attributed to undetected low-Z fragments, whose contribution is not included in the calculation, leading to an overestimated value for the calculated RBE. On the other hand, calculated values 10% greater than the measured RBE are explained as track structure effects due to the higher radial ionization density near neon tracks relative to the ionization density near the silicon tracks used to fit the RBE vs LET infinity data.     

Radioprotection by DMSO of mammalian cells exposed to X-rays and to heavy charged-particle beams

Populations of G1-phase Chinese hamster cells in stirred suspensions containing various concentrations of DMSO were irradiated with 250 kV X-rays or various heavy charged-particle beams. Chemical radioprotection of cell inactivation was observed for all LET values studied. When cell survival data were resolved into linear and quadratic components, the extent and concentration dependence of DMSO protection were found to be different for the two mechanisms. The chemical kinetics of radioprotection for single-events were similar for LET values up to those which gave the maximum RBE. DMSO protected to a lesser extent against energetic argon ions at an median LET of approximately 220 keV/micron. These data could indicate the contribution of indirect action by hydroxyl radicals and hydrogen atoms to cell inactivation by single-hit and double-hit mechanisms for various radiation qualities. The decrease in RBE observed at very high LET may result, in part, from reduced yields of water radicals at 10(-9)-10(-8) s resulting from radical recombination mechanisms within the charged particle tracks.     

Characterization of hydroxyl radical-induced damage after sparsely and densely ionizing irradiation

The extent of hydroxyl radical mediated cell inactivation was measured for a variety of particle beams ranging from 8.5 Me V/u neon ions to 570 Me V/u argon ions. In general, the fraction of the total radiosensitivity caused by OH decreases from close to 60 per cent at low ionization density or low linear energy transfer (low LET) to close to 25 per cent at high LET for aerobically irradiated mammalian cells. The extent of OH induced cell lethality can be explained in terms of LET infinity only for low energy or low atomic number particles where fragmentations and complicated track structures do not contaminate the characteristic particle LET. For example, at a calculated LET infinity of 100 ke V/micron, the OH mediated fraction of the total radiation damage is about 25 per cent for low energy carbon but close to 40 per cent for high energy carbon ions. For low energy charged nuclei of approximately the same energy, as the 5.4-13.4 MeV/u He, Li, C and Ne ions in this report, there is a predictable diminution of the OH mediated effect with increasing LET infinity; however, the biological effect cannot be predicted accurately from calculated LET infinity values for high energy particle irradiation, nor indeed from a variety of low energy charged particles of quite different energies (incident velocities). This illustrates the unsuitability of using LET as a unifying parameter, except under specific circumstances. As more is learned about the energy deposition for energized charged particles in terms of track structure (core and penumbra), it may be possible to characterize the radiobiological data with a better physical parameter than LET infinity.     

Neoplastic cell transformation by heavy ions

We have studied the induction of morphological transformation by heavy ions. Golden hamster embryo cells were irradiated with 95 MeV 14N ions (530 keV/microns), 22 MeV 4He ions (36 keV/microns), and 22 MeV 4He ions with a 100-microns Al absorber (77 keV/microns) which were generated by a cyclotron at the Institute of Physical and Chemical Research in Japan. Colonies were considered to contain neoplastically transformed cells when the cells were densely stacked and made a crisscross pattern. It was shown that the induction of transformation was much more effective with 14N and 4He ions than with gamma or X rays. The relative biological effectiveness (RBE) relative to 60Co gamma rays was 3.3 for 14N ions, 2.4 for 4He ions, and 3.3 for 4He ions with a 100-microns Al absorber. The relationship between RBE and linear energy transfer was qualitatively similar for both cell death and transformation.     

Amplification of the c-myc oncogene in radiation-induced rat skin tumors as a function of linear energy transfer and dose.

The c-myc oncogene was previously shown to be amplified in large, later-stage carcinomas of the rat skin induced by 0.8-MeV electrons. In a panel of 70 tumors induced by neon ions (45 keV/microns), c-myc amplification was rare, and in contrast to the data for tumors induced by low-linear-energy transfer (LET) (0.3 keV/microns) radiation, showed no correlation with tumor size, growth period, or time, but was associated with radiation dose. The tissue specificity for c-myc amplification seen in tumors induced by electrons was not seen in tumors induced by neon ions. These results suggest that quite distinct molecular mechanisms operate even in late stages of carcinogenesis that depend on the LET of the inducing radiation. Furthermore, the results suggest that c-myc amplification observed in tumors induced by low-LET radiation is not a general property of rat skin carcinomas, but is linked mechanistically to the inducing radiation, even though it is not detectable until many months after exposure and tumor appearance.     

Effects of low dose particle radiation to mouse neonatal neurons in culture.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions generated by HIMAC at NIRS and BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis focusing on the dependencies of (1) the animal strains with different radiation sensitivities, and (2) LET with different nuclei. Of the three mouse strains, SCID, B6 and C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to both X-ray and carbon ion ( 290 MeV/n) as evaluated by 10% apoptotic criterion. However, the sensitivity differences among the strains were much smaller in case of carbon ion comparing to that of X-ray. Regarding the LET dependency, the sensitivity was compared with using C3H and B6 cells between the carbon (13 keV/micrometers) and neon (70 keV/micrometers) ions. Carbon (290 MeV/n) did not give a detectable LET dependency from the criterion whereas the neon (400 MeV/n) showed 1.4 fold difference for both C3H and B6 cells. Although a LET dependency was examined by using the most sensitive SCID cells, no significant difference was detected.     

A microdosimetric-kinetic model for the effect of non-Poisson distribution of lethal lesions on the variation of RBE with LET

The microdosimetric-kinetic (MK) model for cell killing by ionizing radiation is summarized. An equation based on the MK model is presented which gives the dependence of the relative biological effectiveness in the limit of zero dose (RBE1) on the linear energy transfer (LET). The relationship coincides with the linear relationship of RBE1 and LET observed for low LET, which is characteristic of a Poisson distribution of lethal lesions among the irradiated cells. It incorporates the effect of deviation from the Poisson distribution at higher LET. This causes RBE1 to be less than indicated by extrapolation of the linear relationship to higher LET, and to pass through a maximum in the range of LET of 50 to 200 keV per micrometer. The relationship is compared with several experimental studies from the literature. It is shown to approximately fit their results with a reasonable choice for the value of a cross-sectional area related to the morphology and ultrastructure of the cell nucleus. The model and the experiments examined indicate that the more sensitive cells are to radiation at low LET, the lower will be the maximum in RBE they attain as LET increases. An equation that portrays the ratio of the sensitivity of a pair of cell types as a function of LET is presented. Implications for radiotherapy with high-LET radiation are discussed.